Bacteria disintegrator



Dec. 13, 1966 w. LJENSEN BACTERIA DISINTEGRATOR Filed Sept. 23, 1963 R O N E W m ATTORNEYS United States Patent 3,291,400 BACTERIA DISINTEGRATOR Wayne I. Jensen, Brigham City, Utah, assignor to the United States of America as represented by the Secretary of the Interior Filed Sept. 23, 1963, Ser. No. 310,957 6 Claims. (Cl. 241-172) The invention herein described and claimed may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of royalties thereon or therefor.

This invention relates to disintegration of microorganisms, particularly bacterial cells for extraction of toxin therefrom.

FIG. 1 is a detail perspective view of the flexible tube that forms a part of the apparatus of the invention.

FIG. 2 is a cross-sectional view of the flexible tube and its contents taken at 2-2 of FIGURE 1.

FIG. 3 illustrates a side elevation view of the complete apparatus used in the invention.

FIG. 4 illustrates a front elevation view of the complete apparatus used in the invention.

In the study of avian botulism, it is desirable to have available for experimental use a standard toxin preparation that is stable with respect to its effects on laboratory animals. Whole cultures of Closrridz'um bo tulirzumare unsatisfactory in that autolysis or mechanical rupture of the bacterial cells occurs during storage at refrigerator or freezing temperatures, gradually releasing quantities of toxin into the medium and thereby increasing its toxicity for test animals. Removal of the bacterial cells in order to stabilize the effective toxin level results in waste of a large part of the toxin content of the culture. For these reasons, a simple, rapid technique is needed for extracting toxin from Clostridium botulinum cells without causing denaturation of the protein molecule by chemical or physical means.

Prior art devices, such as those described by Lamanna, Chatigny and Colledge in J. Bacteriol., vol. 77, January 1959, pp. 104109, have employed a pumping device in which a flexible tube containing a suspension of glass beads and bacteria are moved rapidly over rotating pulleys. However, such devices operating at high 'speed often generate undesirable amounts of heat that may cause undesirable physical or chemical changes in the cell contents. Difficulties in filling and sealing the tube are present as well.

It is therefore an object of the present invention to provide a simple and economic means for disintegrating microorganisms, to obtain toxin, with little chemical or physical change in the cell contents.

It is a further object of the invention to provide an improved apparatus for rupturing cells contained in suspension with a grinding material in a flexible grinding tube. Another object is to provide a simple and effective means for filling the tube.

It has now been found that this objective may be accomplished by the use of the device illustrated in the drawings. A specific embodiment of the invention will therefore be described by reference to the drawings. This specific embodiment is for illustrative purposes only and many modifications may be made as will be subsequently described.

A continuous, circular, flat flexible rubber tube 1 is filled by means of sealed-on side arm 2 with a slurry 3 consisting of a dense water suspension of the bacterial cells and very fine glass beads 3a (about 0.15 mm. average diameter) as the grinding material. This tube is then placed on the flat rim 4 of flywheel 5 which is also provided with flanges 6 and 7.

3,291,400 Patented Dec. 13, 1966 A flat belt 8, running between flywheel 5 and pulley 9 is then positioned over tube 1 on the flywheel. When the flywheel rotates, this belt passes over the surface of tube 1, exerting a gentle massaging action on the bacteriaglass bead mixture.

The rubber tube, containing 40 ml. of a suspension of Clostridium botulinum and an equal volume of glass beads, is of such size as to fit snugly on the rim of the flywheel with an overlap of about inch. The additional inch allows insertion of one end of the tube within the other, thus forming a continuous tube when the ends are sealed together. The ends of the tube and side arms are sealed in place with adhesive and the joints are additionally covered with adhesive tape to ease the tension that they are subjected to when the device is in operation. When the rubber tube has been filled with slurry, and the air thereby forced out, the side arm is plugged with a short section of glass rod. Although the plug is designed to fit snugly, it is sealed in place with adhesive tape as a safety factor.

A notch 10 is provided in flange 7 through which side arm 2 extends. The side arm is then positioned in a short piece of rubber tubing 11, which is secured to the flywheel, to lessen vibration when the wheel is in motion.

Small pulley wheel 9 is mounted on metal plate 13 which is attached to wooden platform 14. The position of platform 14 is adjustable relative to wooden base 12 by means of slots 16 and bolts 15 with wing nuts 15a. This arrangement permits wheel 9 to be moved toward flywheel 5 thus facilitating the placing of the rubber tube and belt in operating position. Tension on the belt (and thus pressure on the rubber tube) is also adjusted by movement of wheel 9 toward or away from the flywheel. In order to keep the flywheel above the level of the wooden base, the bearings 21 which carry the driveshaft are mounted on a block 22.

The device is powered by an electric motor 17 with l /zand 7 /2 inch V-grooved pulley wheels 18 and 19 on the motor shaft 20a and driveshaft 20. With a 1720 rpm. motor an operating speed of approximately 340 rpm. results. This speed can, of course, be modified by suitable changes in the ratio of wheel diameters.

By comparing gram-stained smears of suspensions of Clostridium botulz'num before and after treatment according to the above described process, it has been determined that more than percent of the cells are ruptured within 15 minutes at an operating speed of approximately 340 rpm. Highly toxic extracts have been prepared by rupturing the cells and removing the cell fragments by centrifu gation.

The above description of a specific embodiment of the invention is given for illustrative purposes only; obviously, many modifications will be apparent to those skilled in the art without departing from the essential spirit and scope of the invention as set forth in the claims. For example, a variable speed motor could be used, thus obviating the necessity of changing pulley wheels to modify the operating speed. Also, any convenient means for moving the small wheel could be employed in place of the hinge of the example, or the flywheel could be moved relative to the small wheel.

The grinding tube may be molded from a single piece of rubber or heat-resistant plastic. In addition, the use of two side arms (on opposite sides of the tube) would facilitate flushing out the contents of the tube and help to maintain balance when revolving on the flywheel. Furthermore, the grinding tube may be of any flexible material such as plastic, e.g., Tygon-B.

Other hard, inert materials may be used as the grinding material in place of the glass beads of the example,

e.g., sand, Carborundum, boron carbide, emery, Alundum, porcelain, stainless steel, etc.

The suspending liquid may also be other than water, as employed in the example. Organic liquids may be used and are of particular utility where direct extraction of intracellular consituents is desired.

The invention is also applicable to microorganisms other than the Clostridium botulinum employed in the specific embodiment, e.g., other bacteria, yeasts, etc. Furthermore, the optimum size and amount of grinding material, amount of water in the slurry, type of side arm, size of tubing, size of the flywheel and the smaller wheel, speed of rotation, tension on the belt and length of time of operation of the device may also vary according to the type and amount of bacteria employed. All of these factors are best determined empirically as may be readily accomplished by one skilled in the art.

The method of the invention is particularly advantageous in the case of a toxin, such as the botulinum toxin, where the toxin molecule is easily denatured or inactivated by heat and by intimate admixture with air. This is because 1) due to the relatively low operating speed and short time required to rupture the cells, relatively little heat is generated in the slurry and (2) by exerting pressure on the flexible rubber tube (and thereby decreasing its volume) air can be forced out through the side arm even when small quantities of slurry are used.

What is claimed is:

1. An apparatus for disintegrating microorganisms held in a liquid suspension comprising: (1) a grinding material in a flexible, continuous, closed tube containing said suspension of microorganisms; said tube being mounted on (2) a rotatable flywheel having a flat outer edge and means for containing the tube on said outer edge; (3) a belt mounted over said tube on the flywheel; said belt also extending over (4) a pulley located at a distance from the flywheel suflicient to apply pressure to the flexible tube; (5) means for 'adjustably locating the pulley to adjust the tension on the belt; and (6) means for rotating the flywheel.

2. Claim 1 in which the flexible tube is provided with a side arm for filling the tube with the suspension of microorganisms.

3. Claim 1 in which the liquid is water.

4. Claim 1 in which the grinding material is glass beads.

5. Claim 4 in which the average diameter of the glass beads is about 0.15 mm.

6. Claim 1 in which the flexible tube is rubber.

References Cited by the Examiner UNITED STATES PATENTS 2,650,033 8/1953 Ahl'mann 241-176 XR ROBERT C. RIORDON, primary Examiner.

HARRY F. PEPPER, ]R., Examiner. 

1. AN APPARATUS FOR DISINTEGRATING MICROORGANISMS HELD IN A LIQUID SESPENSION COMPRISING: (1) A GRINDING MATERIAL IN A FLEXIBLE, CONTINUOUS, CLOSED TUBE CONTAINING SAID SUSPENSION OF MICROORGANISMS; SAID TUBE BEING MOUNTED ON (2) A ROTATABLE FLYWHEEL HAVING A FLAT OUTER EDGE; (3) A MEANS FOR CONTAINING THE TUBE ON SAID OUTER EDGE; (3) A BELT MOUNTED OVER SAID TUBE ON THE FLYWHEEL; SAID BELT ALSO EXTENDING OVER (4) A PULLEY LOCATED AT A DISTANCE FROM THE FLYWHEEL SUFFICIENT TO APPLY PRESSURE TO THE FLEXIBLE TUBE; (2) MEANS FOR ADJUSTABLY LOCATING THE PULLEY TO ADJUST THE TENSION ON THE BELT; AND (6) MEANS FOR ROTATING THE FLYWHEEL. 